ES2875853T3 - Method of treating fibrosis - Google Patents

Abstract

A pharmaceutical composition for use in treating a fibrotic condition, the composition comprises: (a) an administrable peptide or polypeptide consisting of the sequence FTTFTVT (SEQ ID NO: 1) and, optionally, wherein the administrable peptide or polypeptide is linked to, or associated with, an internalizing sequence; and (b) a pharmaceutically acceptable carrier or excipient.

Description

description

Method of treating fibrosis

Countryside

The present disclosure is in the field of biochemistry and medicine is directed to methods and compositions to increase the levels of the p53 protein, reduce uPA and uPAR and increase the expression of PAI-1, in fibrotic lung fibroblasts (FL) and reduce its proliferation, and to treat idiopathic pulmonary fibrosis (IPF) using nutlin-3a and peptides.

Description of the technical background

Idiopathic pulmonary fibrosis (IPF) is a poorly understood progressive and fatal lung disease for which there is no treatment other than lung transplantation (Mason DP et al., Ann Thorac Surg 84.í 121-8, 2007). The 5-year median survival after diagnosis is less than 20%. Most forms of interstitial lung diseases and other forms of pulmonary fibrosis are characterized by fibrotic lesions, progressive distortion of the alveolar architecture and replacement with fibrotic or scar tissues with excess deposit of extracellular matrix (ECM) (American Thoracic Society, Am J Respir Crit Care Med 161: 646-664, 2000; Noble PW et al., Clin Chest Med 25: 749-758, 2004; Selman M et al., Ann Intern Med 134: 136-151, 2001) . This produces progressive dyspnea and loss of lung function. A distinctive morphological lesion is the spatial and temporal heterogeneity that incorporates areas of normal lung that are directly adjacent to areas of fully established fibrosis, microscopic honeycombing, and areas of evolving fibrosis that contain proliferating, collagen-producing fibroblasts / myofibroblasts, the so-called "Fibrotic foci".

IPF is the most common chronic, progressive, and fatal interstitial lung disease of unknown etiology with an estimated incidence of 40-50 cases per 100,000 individuals in the United States. The viability, activation of pulmonary fibrotic fibroblasts ("FL") (or myofibroblasts), production and deposition of ECM typify the IPF lungs (Selman M et al., Expert Opin Emerg Drugs 16: 341-62, 20l1; Shetty, S et al. Am J Respir Cell Mol Biol i 5: 78-87, 1996; Zhu S et al., Am J Physiol: Lung Cell Mot Physiol 297.L97-108, 2009; Suganuma H et al., Thorax 50: 984- 9, 1995; American Thoracic Society, supra; Noble PW et al., Supra).

Previous work by the present inventors (and others) showed that pulmonary fibroblasts including FL fibroblasts from the lungs of IPF patients express urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), and plasminogen activator inhibitor 1 (PAI). -1) (Shetty et al., 1996, supra; Shetty S and Idell S. Am J Physiol 274: L871-L882, 1998; Chang W et al., J Biol Chem 285: 8i 96-206, 2010). uPA is mitogenic for both normal lung fibroblasts (NL) and FL, and the process involves the binding of uPA to uPAR through the growth factor domain of uPA (Tkachuk V et al. Clin Exp Pharmacol Physiol 23: 759-65, 1996; Padró T et al., J Cell Sci 115.í 961-71, 2002; Shetty S et al., Am J Physiol 268: L972-L982, 1995; Shetty S et al., Antisense Res Dev 5: 307- 314, 1995). Furthermore, uPA increases uPAR expression (Shetty S et al., J Biol Chem 276: 24549-56, 2001; Shetty S et al., Am J Respir Cell Mol Biol 30: 69-75, 2004). Several years ago, the present inventors reported that FPI lung FL fibroblasts express significantly more uPA and uPAR, and show a higher rate of basal and uPA-mediated proliferation than NL fibroblasts (Shetty et al., 1996, supra; 1998, supra ). Other groups confirmed that increased uPAR expression by FL fibroblasts from IPF patients contributes to migratory behavior (Mace KA et al., J Cell Sci 118: 2567-77, 2005; Basire A et al., Thromb Haemost 95: 678- 88, 2006; Zhu, S. et al., 2009, supra

Studies by the present inventor and co-workers found that uPA regulates epithelial cell apoptosis / survival by regulating p53 (Shetty S et al., 2005, supra) which controls reciprocal expression of uPA (Shetty P et al., Am J Resp Cell Mol Biol, 39: 364-72, 2008), its uPAR receptor (Shetty S et al. Mol Cell Biol 27: 5607-5618,2o07) and its main inhibitor PAI-1 (Shetty S et al. J Biol. Chem 283: 19570-80, 2008) at the post-transcriptional level and involves a novel cell surface signaling interaction between uPA, uPAR, caveolin-1 ("Cav-1") and p-lintegrin (Shetty S et al., 2005, supra) . Based on an appreciation of the foregoing, the present inventors devised new compositions and methods for treating ALI and its consequent remodeling reactions.

During lung fibrosis (a term used interchangeably with “pulmonary” fibrosis), the expression of the transcription factor p53, known primarily as a tumor suppressor protein, is severely suppressed in fibrotic fibroblasts which in turn induces expression of uPA and uPAR while the expression of PAI-1 is significantly inhibited. Suppression of PAI-1 expression and concurrent induction of uPA and uPAR expression as a consequence of inhibition of p53 expression in fibrotic fibroblasts results in fibroblast proliferation and ECM deposition, ie, fibrosis. The increased interaction of mdm2 with p53 and subsequent mdm2-mediated ubiquitination of p53 contributes to the inhibition of p53 in fibrotic fibroblasts.

A reciprocal relationship between the activities of p53 and NF-kB in cancer cells has been demonstrated, but there is little information regarding interactions between p53 and NF-kB in inflammatory processes. Liu G et al. ( J Immunol. 182: 5063-71 (2009)) found that neutrophils and macrophages lacking p53 (p53 ('/') have greater responses to stimulation with bacterial lipopolysaccharide (LPS) than p53 (+ / +) cells, and they produce higher amounts of pro-inflammatory cytokines, including TNF- ^ci , IL-6 and MIP-2, and demonstrated binding activity to A ^d N of NF- ^k B increased. P53 ('/ _) mice are more susceptible than p53 (+ / +) mice to LPS-induced acute lung injury (ALI). The increased response of p53 (+ / +) cells to LPS does not involve alterations in intracellular signaling events associated with the involvement of the toll-like receptor TLR4 (eg, activation of MAP kinases, phosphorylation of IkB-ci or the p65 subunit of NF-kB, or degradation of IkB-ci. Culture of neutrophils and macrophages stimulated with LPS with nutlin-3a, attenuated the DNA-binding activity of NF-kB and the production of pro-inflammatory cytokines. Treatment of mice with nutlin -3a reduced the severity of LPS-induced ALI The authors concluded that p53 regulates NF-kB activity in inflammatory cells and suggested that modulation of p53 may have potential therapeutic benefits in acute inflammatory conditions such as ALI.

Nutlina (or nutlin-3a) (also abbreviated NTL)

The chemical structure of the molecule 4-2-4,5-dihydro-1H-imidazol-1-piperazin-2-one (C30H30CI2N4O4) also called nutlin or nutlin-3a is as shown below

Formula 1

NTL is an antagonist of MDM2, which is an activator of p53 and an inducer of apoptosis. MDM2 works by binding to the tumor suppressor protein p53 and downregulating its transcriptional activity and stability. Inhibition of the MDM-p53 interaction results in stabilization of p53, cell cycle arrest, and apoptosis. NutIin-3 has shown potential as a cancer treatment by activating the p53 pathway in cancer cells (Tovar, C. et al., Prnc Nati Acad Sci USA 103: 1888-1893 (2006); Vassilev, LT et al. Science 303 844-848 (2004); El-Deiry, WS Cancer J 11: 229-236 (1998)).

Document U.S. 7,893,278 (Haley et al.) Disclosed chiral nutIin-3 both generically and specifically. Document U.S. 6,734,302 for racemic nutIin-3. The '278 patent teaches a compound of the formula

Formula 2

which is described further below.

Both of the above patents discuss uses as inhibitors of the Mdm2-p53 interaction and in cancer treatment.

US Patent Publication 2011/0301142 (Hutchinson et al.) Teaches a method of treating idiopathic pulmonary fibrosis in a mammal which comprises administering a therapeutically effective amount of an LPA1 receptor antagonist to the mammal. The antagonist can be certain imidazole derivatives, but not imidazoline-like compounds such as NTL.

US 6,596,744 (Wagle et al.) Discloses a method of treating or ameliorating certain fibrotic diseases with heterocyclic compounds, all of which are clearly other than NTL. Reported diseases include Fibrotic lung diseases that have as a manifestation fibrotic hypertrophy or fibrosis of lung tissue. These diseases include pulmonary fibrosis (or interstitial lung disease or interstitial pulmonary fibrosis), idiopathic pulmonary fibrosis, the fibrotic element of pneumoconiosis, pulmonary sarcoidosis, fibrosing alveolitis, the fibrotic or hypertrophic element of cystic fibrosis, chronic obstructive pulmonary disease, respiratory distress syndrome adult and emphysema.

Dey et al. in Cell Cycle 6:17, 2178-2185 (2007) describes that nutlins were identified as the first potent and specific small molecule Mdm2 antagonists that inhibit the p53-MDM2 interaction.

None of the above documents disclose treating IPF with nutlin-3a.

Caveolin-1 Derived Peptides

The present inventors discovered that a 20 residue peptide DGIWKASFTTFTVTKYWFYR, SEQ ID NO: 2) which is the scaffold domain of caveolin-1 (Cav-1; SEQ ID NO: 3, shown below) protected pulmonary epithelial cells (LEC) from Bleomycin-induced apoptosis ("BLM") in vitro and in vivo and prevented subsequent pulmonary fibrosis by attenuating epithelial lung damage (Shetty et al., allowed US patent application 12 / 398,757 published as document US 2009-0227515A1 (10 Sept., 20o9).

The present inventors also found a 17-residue peptide NYHYLESSMTALYTLGH (SEQ ID NO: 4), designated PP-2, also protected LEC from BLM-induced apoptosis in vitro and in vivo and prevented subsequent lung fibrosis by attenuating epithelial lung damage (Shetty et al., supra).

Shetty et al., 2009 ( supra) also describe biologically active substitution, addition and deletion variants of these peptides, as well as administrable peptide and polypeptide multimers comprising the above peptides, and pharmaceutical compositions comprising the above peptides, variants and multimers . Those compositions inhibit apoptosis of injured or damaged lung epithelial cells and treat acute lung injury and consequent pulmonary fibrosis / IPF.

The above document did not identify a particular CSP fragment (later disclosed as part of the present invention) called CSP-4, which has the sequence FTTDTVT (SEQ ID NO: 1) and which has the biological activity of CSP and constitutes part of the object of the present invention.

Caveolin-1 derived peptides are further disclosed in WO 2013/184482 A1; E. Tourkina ET AL: "Antifibrotic properties of caveolin-1 scaffolding domain in vitro and in vivo", American journal of physiology. Lung cellular and molecular physiology, vol. 294, no. 5, 18 January 2008 (18-01-2008), pages 843-861; US 2009/075875 A1; ARBUZOVA, A. ET AL .: "Membrane Binding of Peptides Containing Both Basic and Aromatic Residues. Experimental Studies with Peptides Corresponding to the Scaffolding Region of Caveolin and the Effector Region of MARCKS", BIOCHEMISTRY, vol. 39, no. 33, 2000, pages 10330-; WANASKI, S. P. ET AL: "Caveolin Scaffolding Region and the Membrane Binding Region of Src Form Lateral Membrane Domains", BIOCHEMISTRY, vol. 42, no. 1,2003, pages 42-56; HORTON, M. R. ET AL: "Phase behavior and the partitioning of caveolin-1 scaffolding domain peptides in model lipid bilayers", JOURNAL OF COLLOID AND INTERFACE SCIENCE, vol. 304, no. 1,2006, pages 67-76; GUO, C. -J. ET Al.: "Involvement of caveolin-1 in the Jak-Stat signaling pathway and infectious spleen and kidney necrosis virus infection in mandarin fish (Siniperca chuatsi", Mo LECULAR IMMUNOLOGY, vol. 48, no. 8, 2011, pages 992- 1000; DE LA TORRE, BG ET AL .: "On choosing the right ether for peptide precipitation after acid cleavage", JOURNAL OF PEPTIDE SCIENCE, vol. 14, no. 3, 2008, pages 360-363; EPAND, RM ET AL .: "Caveolin Scaffolding Region and Cholesterol-rich Domains in Membranes", JOURNAL OF MOLECULAR BIOLOGY, vol. 345, no. 2, 2004, pages 339-350; SHETTY, SK ET AL .: "Regulation of Airway and Alveolar Epithelial Cell Apoptosis by p53-Induced Plasminogen Activator Inhibitor-1 during Cigarette Smoke Exposure Injury ", AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY, vol. 47, no. 4, 2012, pages 474-83; document WO 01/11038 A2; or document WO 2009/111625 A2.

In view of the poor prognosis and lack of therapeutic approaches for FPl, there is an urgent need for new interventions to reverse or at least slow the progression of the disease. This critical therapeutic gap is addressed by the present invention.

This disclosure is an extension of the inventor's previous findings (S. Shetty et al., 2007, 2008 & 2009, supra). As described in subsequent sections, p53 regulates the expression of uPA, uPAR, and PAl-1. The process involves MDM2-mediated control of p53 protein expression at the post-translational level without affecting p53 mRNA. These observations demonstrate a causal link between p53 expression and the uPA fibrinolytic system in fibrotic repair.

Compendium

Inhibition of the interaction of p53 protein with MDM2 using NTL increases p53 and PAl-1 while inhibiting uPA and uPAR. These changes produce apoptosis of fibrotic fibroblasts and inhibition of fibroblast proliferation. fibrotic cells isolated from the lungs of patients with IPF, as well as from the lungs of mice with BLM-induced accelerated pulmonary fibrosis.

According to the present invention, NTL inhibits established and progressive pulmonary fibrosis by inducing or increasing the expression of p53 in fibrotic fibroblasts. Therefore, this compound is useful for treating pulmonary fibrosis in a subject in need thereof.

The present inventors envisioned that impaired p53-mediated control of the uPA fibrinolytic system in FL fibroblasts is a basis for targeted IPF therapy. In FL fibroblasts, the basal expression of p53 and microRNA-34a (miR-34a) are markedly suppressed while the levels of ECM proteins such as collagen-I (CoI-I) and smooth muscle actin (ci-SMA) are augmented. Treatment of FL fibroblasts with the mdm2 inhibitor compound nutlin-3a or CSP-4 increases the expression of p53 and PAI-1 with reciprocal inhibition of uPA and uPAR. Treatment of FL fibroblasts with nutlin-3a or Cs P-4 also inhibits ECM production. These preliminary findings and recent publications by the present inventors and co-workers justify an interventional approach to reestablish the interrelation between p53 and the fibrinolytic system of uPA which is otherwise distorted in FL fibroblasts resulting in pulmonary fibrosis. p53 has pleiotropic effects beyond the control of the uPA2 fibrinolytic system. However, PAI-1 is the downstream effector of p53 in fibroblasts and other cells.

Importantly, the reestablishment of p53 in FL fibroblasts modulates uPA, uPAR, and PAI-1 expression, cell viability, and ECM production. The p53-fibrinolytic system targeting approach of uPA in FL fibroblasts is illustrated in Figure 1. p53 induces miR-34a transcription while miR-34a increases p53 acetylation by inhibiting histone deacetylase sirtuin 1 (SIRT1) . This leads to the stabilization of the p53 protein due to the inhibition of its degradation mediated by mdm2. However, in FL fibroblasts, basal levels of p53 and miR-34a protein are markedly suppressed due to increased ubiquitination of p53 protein by mdm2 and as a consequence of low basal expression of caveolin-1. This leads to reduced p53-mediated inhibition of uPA and uPAR, or concurrent induction of PAI-1. These changes contribute to the excessive proliferation of FL fibroblasts and ECM production and are reversed by CSP-4 and nutilin-3a. CSP-4 and nutilin-3a increase p53 levels by inhibiting mdm2-mediated degradation of the p53 protein. Restoration of p53 expression and p53-mediated changes in the uPA fibrinolytic system in FL fibroblasts results in attenuation of viability, and restricts ECM production and deposition. Fibrosis is inhibited in BLM-exposed mice with established pulmonary fibrosis through the restoration of p53 levels in FL fibroblasts which in turn suppresses the uPA and uPAR proliferation signals while inducing the pro-apoptotic signal for these cells; PAI-1.

The inventors show, for the first time, that p53-mediated changes of the uPA fibrinolytic system regulate the profibrotic response of pulmonary fibroblasts that are central to the pathogenesis of pulmonary fibrosis and that this route may be targeted for therapeutic benefit. Unlike histologically “normal” lung NL fibroblasts, FL fibroblasts, including fibroblasts / myofibroblasts from fibrotic lung tissues, express very low levels of baseline p53, miR-34a, and caveolin-1 whereas uPA and uPAR expressions are high. Treatment of FL fibroblasts with CSP-4 or the low molecular weight compound nutilin-3a restores the expression of p53 and miR-34a, and induces PAI-1. These changes suppress uPA and uPAR expression and inhibit MEC deposition. These agents also reverse BLM-induced pulmonary fibrosis and protect the lung epithelium, also demonstrating an interrelation between p53 and regulation of the fibrinolytic system. Thus, CSP-4-mediated or nutilin-3a-mediated mitigation of pulmonary fibrosis represents novel therapeutic approaches.

There is currently no effective treatment to reverse pulmonary fibrosis. The present disclosure addresses this important gap and provides novel compounds and methods for targeting FL fibroblasts and interplay between p53 and the uPA fibrinolytic system to effectively treat fibrotic lung disease.

The invention is defined by the appended claims.

Specifically, the present disclosure is directed to a method of increasing p53 protein levels, reducing uPA and uPAR, and increasing PAI-1 expression in fibrotic lung fibroblasts (FL), which comprises providing FL fibroblasts with an effective amount of a compound that inhibits the interaction of MDM2 with p53 protein and MDM2-mediated degradation of p53, wherein the compound is:

(a) NTL or a chiral cis-imidazoline analog of NTL that inhibits the MDM2-p53 interaction;

(b) a peptide selected from the group consisting of:

(i) CSP-4, the sequence of which is FTTFTVT (SEQ ID NO: 1);

(ii) an addition variant of the CSP-4 addition variant peptide that does not exceed 20 amino acids in length; (iii) a covalently modified chemical derivative of the CSP-4 peptide, or

(c) a peptide multimer comprising at least two monomers each monomer is the CSP-4 peptide, the variant or the chemical derivative of (b),

wherein the NTL analog, peptide variant, peptide chemical derivative, or peptide multimer has at least 20% of the biological ^or biochemical activity of NTL ^or the CSP-4 peptide in an in vitro ^or in ^vivo assay.

The NTL analog, peptide variant, peptide chemical derivative, or peptide multimer described above or below has the following activity relative to the activity of NTL or CSP-4, respectively: at least about 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, approximately 95%, 97%, 99%, and any drivable interval therein, such as, by example, from about 70% to about 80%, and more preferably from about 81% to about 90%; or even more preferably, from about 91% to about 99%. This relative activity can be based on any method disclosed herein or known in the art for evaluating such activity.

In a preferred embodiment of the above method, the compound is NTL. In another embodiment, the compound is the CSP-4 peptide of SEQ ID NO: 1. In another embodiment of the method, the compound is the peptide multimer, preferably one comprising CSP-4 peptide monomers (SEQ ID NO: 1).

Preferably, in the above method using a peptide multimer

(a) the peptide multimer has the formula P1n where

(i) P1 is the ^{chemical peptide, variant or} derivative; and

(ii) n = 2-5, or

(b) the peptide multimer has the formula (P1-Xm) n-P2, where

(i) each of P1 and P2 is, independently, the ^{chemical peptide, variant or} derivative;

(ii) each of P1 and P2 is the same ^or different peptide, variant ^or chemical derivative;

(iii) X is C1-C5 alkyl, C1-C5 alkenyl, C1-C5 alkynyl, C1-C5 polyether containing up to 4 oxygen atoms;

(I ^v ) m = 0 ^or 1; and

(v) n = 1-7, or

( ^c ) the peptide multimer has the formula (P1-Glyz) n-P2, where:

(i) each of P1 and P2 is, independently, the peptide, variant ^or derivative,

(ii) each of P1 and P2 is the same ^or different peptide, variant, ^or derivative;

(iii) ^z = 0-6; and

(Iv) n = 1-25,

Preferably the multimer has at least 20% of the biological activity of the CSP-4 peptide in an in vitro or in vivo assay.

Also disclosed is a method of treating a mammalian subject, preferably a human, having a disease or condition characterized by pulmonary fibrosis (i.e., IPF), which comprises administering to the subject an effective amount of a compound that, by inhibiting degradation MDM2-mediated p53 protein, increases p53 protein levels, reduces uPA and uPAR levels, and increases PAI-1 expression in FL fibroblasts. In a preferred embodiment of this method, the compound is

(a) Nutilin-3a (NTL) or a chiral cis-imidazoline analog thereof that inhibits the MDM2-p53 interaction;

(b) a peptide selected from the group consisting of:

(i) CSP-4, the sequence of which is Ft TFTVT (SEQ ID NO: 1), the most preferred peptide embodiment; (ii) an addition variant of the CSP-4 addition variant peptide that does not exceed 20 amino acids in length; (iii) a covalently modified chemical derivative of the CSP-4 peptide, or

(c) a peptide multimer comprising at least two monomers each monomer is the CSP-4 peptide, the variant or the chemical derivative of (b), most preferably a CSP-4 multimer,

wherein the analog, peptide variant, peptide derivative, ^or peptide multimer has at least 20% of the biological or biochemical activity of NTL or c-peptide SP-4 in an in vitro or in vivo assay.

In this method, the compound is preferably in a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier or excipient. The compound is preferably a pharmaceutically acceptable salt of Nt L, NTL analog or peptide compound. In a preferred embodiment of the method, the compound is NTL. In another preferred embodiment of the method, the compound is the peptide multimer, most preferably a multimer comprising CSP-4 FTTFTVT peptide monomers (SEQ ID NO: 1).

The present disclosure is also directed to compositions of matter. In one embodiment the composition is a peptide that increases p53 protein levels, reduces uPA and increases PAI-1 expression in FL fibroblasts, preferably selected from the group consisting of:

(a) a peptide designated CSP-4 whose sequence is FTTFTVT (SEQ ID NO: 1),

(b) an addition variant of the CSP-4 peptide up to about 20 amino acids in length and not the scaffold domain (CSP) of caveolin-1 (Cav-1) having the sequence SEQ ID NO: 3;

(c) a covalently modified chemical derivative of the CSP-4 peptide of (a),

(d) a covalently modified chemical derivative of the variant of (b),

whose variant ^or chemical derivative has at least 20% of the biological ^or biochemical activity of the CSP-4 peptide in an in vitro or in vivo assay. The chemical variant or derivative may have

A preferred composition is the heptapeptide designated CSP-4, FTTFTVT (SEQ ID NO: 1).

Another composition is a peptide multimer that comprises at least two monomers, each monomer is the CSP-4 peptide, the variant or the chemical derivative, whose multimer:

(a) has the formula P1n where

(i) P1 is the peptide, variant or chemical derivative as above, and

(ii) n = 2-5, or

(b) has the formula (P1-Xm) n-P2, where

(i) each of P1 and P2 is, independently, the peptide, variant, or chemical derivative as above, (ii) each of P1 and P2 is the same or different peptide, variant, or derivative

(iii) X is C1-C5 alkyl, C1-C5 alkenyl, C1-C5 alkynyl, C1-C5 polyether containing up to 4 oxygen atoms;

( ^iv ) m = 0 ^or 1; and

(v) n = 1-7,

(c) has the formula (P1-Glyz) n-P2, where:

(i) each of P1 and P2 is, independently, the peptide, variant or derivative,

(ii) each of P1 and P2 is the same or different peptide, variant, or derivative;

(iii) ^z = 0-6; and

(iv) n = 1-25,

wherein the peptide multimer has at least 20% of the biological activity of the CSP-4 peptide in an in vitro or in vivo assay.

the following CSP-4 related activity: at least about 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, about 95% , 97%, 99%, and any range derivable therein, such as, for example, from about 70% to about 80%, and more preferably from about 81% to about 90%; or even more preferably, from about 91% to about 99%.

Also provided is an antifibrotic pharmaceutical composition comprising:

(a) the above peptide variant or chemical derivative; and

(b) a pharmaceutically acceptable carrier or excipient.

Most preferred is the above pharmaceutical composition formulated for injection or pulmonary instillation.

The present disclosure is also directed to the use of NTL, the above NTL analog, the above peptide, variant, chemical derivative, or multimer for use in increasing p53 protein levels, reducing uPA, and increasing PAI-1 expression in FL fibroblasts. , or in treating a disease or condition characterized as pulmonary fibrosis.

Also disclosed is the use of NTL, the above NTL analog, the above peptide, variant, chemical derivative or peptide multimer as defined above for the manufacture of a medicament for use in treating a subject having a disease or condition characterized as pulmonary fibrosis.

Brief description of the drawings

Figure 1 is a schematic illustration showing that low basal p53 expression and disturbed p53-mediated control of the uPA fibrinolytic system in fibrotic lung fibroblasts (FL) produces fibrogenesis. The mechanisms of action and pathways involved in IPF and its treatment by the methods and compositions of the present invention are outlined.

Figures 2A-2I. Fibrotic foci in lungs FPI inuida show decs expression of p53 in fibroblasts FL. Lung sections from IPF patients and "normal" control subjects were stained for H&E (Fig. 2A), vimentin (Fig. 2B), trichrome (Fig. 2C), p53 (Fig. 2D), PAI-1 (Fig. 2E), Kí-67 (Fig. 2F), immunofluorescence for PAI and SP-C (Fig. 2G) or p53 and SP-C (Fig. 2H) or p53 and PAI-1 (Fig. 21). A representative example is shown (n = 5 fpi lung samples).

Figures 3A-3B. Caveolin-1, p53 and PAI-1 are decreased and uPA is increased in human lung fibroblasts FL (IPF) IPF is associated with increased coI-I and inhibition of miR-34a. (Fig. 3A) Cell lysates from normal lung (NL) and FL fibroblasts were immunoblotted for changes in proteins. The figure illustrates typical results for NL fibroblasts (n = 10 cell lines) and FL fibroblasts (n = 18 cell lines). (Fig. 3B) Total RNA from fibroblasts NL (n = 3) and FL (n = 4) was assayed for miR-34a expression by real-time PCR. Northern blots of RNA from representative samples are shown using a 32P-labeled antisense probe for miR-34a and the npRNA loading control (U6) at the bottom of the bar.

Figures 4A-4B. Ios dispar expression of uPA mRNA, uPAR and PAI-1 by fibroblasts lungs FPI and "normal". Total RNA isolated from lung tissues of "normal" subjects and patients with IPF (Fig. 4A) or from fibroblasts NL (n = 6) and FL (n = 8) (Fig. 4B) for uPA mRNA, pA | -1 and coI-I by Pc R in quantitative real time ((RT-PCR) and normalized to the corresponding levels of p-actin mRNA.

Figures 5A-5D. Differential expression of uPA and PAI-1 by FL fibroblasts versus NL from mice. FL fibroblasts isolated from mouse lungs 21 days after injury with BLM or NL fibroblasts from control mice exposed to intranasal saline are described elsewhere (Basnhary et al., 2012, supra). Fibroblast lysates NL and FL were immunoblotted for changes in the expression of p53, uPA, PAI-1 and coI-I proteins (Fig. 5A). Total RNA extracted from mouse lung tissues (Fig. 5B) or from NL and FL fibroblasts (n = 6) (Fig. 5C) from lung tissues of mice with BLM-induced fibrosis or control mice (saline) was assayed for UPA, PAI-1 and collagen-I (CoI-I) mRNA by quantitative real-time PCR. Mouse NL and FL fibroblasts were grown in 12-well plates in DMEM medium. After three days the fibroblasts were counted to determine the proliferation rate (Fig. 5D).

Figures 6A-6D. CSP (CSP4) increases p53 expression by inducing miR-34a in FL fibroblasts (FPI). (Fig. 6A) NL and FL fibroblasts (n = 3) were treated with PBS, CSP-4 (CSP) or control peptide (CP). RNA for miR-34a was analyzed by real-time PCR. (Fig. 6B) FL fibroblasts were treated with PBS or CSP or CP with or without antisense miR-34a (miR-34a-AS) or pre-miR-34a or control miRNA (miR-Ctr). Conditioned media (CM) for PAl-1 and lysates (CL) for p53 and p-actin were immunoblotted. (Fig. 6C) FL fibroblasts were treated with PBS or CSP or CP in the presence or absence of miR-34a-AS or pre-miR-34a or miR-Ctr. RNA was analyzed for miR-34a changes by real-time PCR. (Fig. 6D) A series of overlapping deletions were made in CSP and these peptides were used to treat FL fibroblasts with. Changes in p53, uPA, PAl-1 and coI-I were then evaluated to identify the minimum amino acids required for the effects of CSp.

Figures 7A-7B. Increased mdm2-mediated p53 protein degradation in FL fibroblasts (IPF). (Fig. 7A) NL and FL fibroblast lysates (n = 3) were immunoblotted for p53, mdm2 and p-actin. (Fig. 7B) NL and FL fibroblast lysates were immunoprecipitated (IP) with anti-mdm2 antibody and immunoblotted (IB) for the associated p53 protein.

Figures 8A-8C. Role of the p53-fibrino lytic system interrelation in nutlin-3a (NTL) ^or CSP-4 mediated inhibition of coI-I expression in FL fibroblasts (FPI). (Fig. 8A) FL fibroblasts were exposed to PBS, nutlin-3a (10 mM), CSP-4 (10 nM), or control peptide (CP) for 48 hours. Cell lysates were immunoblotted for the expression of CoI-I, PAl-1, p53, uPA, and p-actin. (Fig. 8B) FL fibroblasts grown in 12-well plates in DMEM medium were exposed to vehicle (PBS) or nutlin-3a, CSP-4 or CP. After 3 d, the cells were separated and counted. (Fig. 8C) FL fibroblasts were transduced with either empty adenovirus vector alone (Ad-EV) or Ad vector expressing p53 (Ad-p53), PAl-1 (Ad-PAI-1) or caveolin-1 (Ad-Cav -1). After 48 h, the cell lysates were immunoblotted for CoI-I, PAl-1, p53, upA and p-actin.

Figures 9A-9B. Role of the p53-fibrino lytic system interrelation in the induction of coI-I expression in NL fibroblasts. NL fibroblasts isolated from histologically "normal" human lungs were treated with p53 shRNA in lentiviral vector to inhibit basal p53 expression. Control cells were treated with non-specific shRNA (Ctr). (Fig. 9A): Conditioned media samples were immunoblotted for PAl-1, uPA, and soluble coI-I while cell lysates were assayed for p53, (a-SMA) and p-actin. (Fig. 8B): Total RNA isolated from Nl fibroblasts treated with control or p53 shRNA was analyzed for changes in mRNA expression of uPA, PAl-1, coI-I and p-actin by quantitative real-time PCR.

Figures 10A-10D. Nutlin-3a (NTL) inhibits pulmonary fibrosis in BLM-treated mice. Mice were exposed to BLM for 14 d to induce pulmonary fibrosis. Saline-treated mice were used as fibrosis controls. After 14 d, BLM-exposed mice were injected IV with vehicle or nutlin-3a (10 mg / kg body weight) (Zhang F et al. Drug Metab Dispos 39.i 5-21, 2011) to determine whether nutlin -3a inhibited established pulmonary fibrosis. On day 21 after BLM injury, mice underwent CT to assess the extent of fibrosis (Fig. 10A). A representative example is shown (n = 9). (Fig. 10B) Lung compliance and resistance were measured in the same mice using the Flexivent system to assess improvement in lung function after nutlin-3a treatment (n = 9). (Fig. 10C) Pulmonary sections were subjected to trichrome staining to assess lung architecture and collagen deposition as an indication of pulmonary fibrosis. (Fig. 10D) Whole lung homogenates were analyzed for total collagen (hydroxyproline) and desmosine content as an independent assessment of changes in ECM.

Figures 11A-11D. CSP-4 inhibits established pulmonary fibrosis and improves lung function. Mice were challenged with BLM to induce pulmonary fibrosis. After 14 d, the mice were injected IV with vehicle or 1.5 mg / kg body weight of CSP-4 or mixed sequence control peptide ("CP"). On day 21 after BLM injury, the mice underwent t Ac to assess the extent of fibrosis (Fig. 11A). A representative example is shown (n = 9). (Fig. 11B): Lung volumes were measured in the same mice (n = 9) using quantitative CT renditions. (Fig. 11C): Lung sections were subjected to trichrome staining and H&E staining (not shown) to evaluate collagen deposition as an indicator of pulmonary fibrosis. (Fig. 11D) Analysis of whole lung homogenates for total hydroxyproline content.

Figures 12A-12C. CSP-4 or nutlin-3a (NTL) inhibits the proliferation of FL fibroblasts. Mice were challenged with BLM to induce significant pulmonary fibrosis or with saline (fibrosis controls). 14 d later, BLM challenged mice were treated with CSP-4, CP or nutlin-3a (see Figures 10A0D / 11A-D). (Fig. 12A): Lung sections (d21 after BLM injury) were subjected to immunohistochemistry (IHC) for Ki-67 to assess proliferation. A representative example is shown (n = 9). Fibroblasts were isolated from the lungs of mice exposed to saline, BLM or BML + nutlin-3a (as described in Figure 11A). Changes in the expression of proteins coI-I, p53 and later uPA and PAI-1 were tested. by immunoblotting (Fig. 12B); and mRNA by quantitative RT-PCR (Fig. 12C).

Figure 13. Inhibition of BLM-induced pulmonary fibrosis by ex vivo treatment of lung tissues of WT mice with CSP-4 or nutlin-3a (NTL). Mice were exposed to intranasal BLM for 21 d to induce pulmonary fibrosis. These mice (n = 3) were sacrificed, the lungs were excised, cut into small pieces, and plated with DMEM medium containing 10% fetal calf serum. The tissue samples were subsequently treated with CSP-4 (10 nM), control peptide (CP) and nutlin-3a (NTL) (10 µM) for 72 h. Conditioned media and tissue lysates were prepared and analyzed for changes in coI-I, α-SMA, and p-actin by immunoblotting.

Description of preferred embodiments

The present inventors found that the basal expression of the "tumor suppressor" protein, p53 and plasminogen activator inhibitor-1 (PAI-1) are markedly reduced in FL fibroblasts obtained from the lungs of f P i patients. This is also true for mice with BLM-induced accelerated pulmonary fibrosis, an accepted model of human IPF. p53 regulates the expression of major components of the uPA fibrinolytic system (uPA, uPAR, and PAI-1). However, it is not clear how changes in the levels of these proteins contribute to the pathogenesis of pulmonary fibrosis, and whether the restoration of the normal mode of the p53-fibrinolytic system interface of uPA mitigates pulmonary fibrosis.

According to the present invention, increased expression of uPA and uPAR, and a concurrent inhibition of PAI-1 due to a lack of expression of p53 in FL fibroblasts are critical for the development of pulmonary fibrosis. Induction of p53 expression, and reestablishment of the p53-uPA fibrinolytic system interplay through p53-mediated reduction of uPA, and a concurrent increase in PAI-1 expression in FL fibroblasts, mitigates IPF and is the basis for treating this disease with NTL.

Unlike normal lung ("NL") fibroblasts, fibroblasts obtained from the lungs of IPF patients ("FL" fibroblasts) or BLM-induced pulmonary fibrosis mice express minimal p53. Furthermore, FL fibroblasts produce low levels of PAI-1, while the expression of uPA and uPAR are markedly elevated.

The present invention addresses the three main components (uPA, uPAR and PAI-1) of the uPA fibrinolytic system in a coordinated and comprehensive manner. Targeting all three major components of the uPA fibrinolytic system at the same time by restoring p53 expression in FL fibroblasts mitigates fibrosis.

Accordingly, this treatment mainly affects FL fibroblasts and lung tissue isolated from IPF patients ex vivo and in vivo. These changes were observed in a mouse model with established pulmonary fibrosis in which the intermediates and subsequent mediators of the beneficial effects of nutlin-3a were defined.

Nutlina-3a

Compounds useful in the method of the present disclosure include nutlin-3a (also called nutlin and NTL) which is 4-2-4,5-dihydro-1H-imidazol-1-piperazin-2-one (C30H30CI2N4O4)

This compound is described in US 7,893,278 (Haley et al). Also useful in the present disclosure are a broader genus of chiral cis-imidazoline analogs of NTL that share the property of inhibiting MDM2-p53 interactions. By NTL analogs is intended the genus of molecules disclosed in the '278 patent, and characterized by formula 2 below.

Formula 2

in which

^{R is selected from saturated or} unsaturated 5- and 6-membered rings containing at least one heteroatom; the heteroatom can be S, N or O. The ring can be substituted with a lower alkyl group which can be further substituted with at least one of -C = OR ⁱ , -SO2CH3, ^or -CH2C = OCH3,

R ⁱ is selected from the group consisting of H, -NH2, -N-lower alkyl, hydroxy-substituted lower alkyl, -NH2-substituted lower alkyl, and a saturated 5- ^or 6-membered ring containing at least one heteroatom (S , N or O);

X ⁱ and X2 are independently selected from the group consisting of H, lower alkoxy, -CH2OCH3, -CH2OCH2CH3, -OCH2CF3, and -OCH2CH2F,

Y ⁱ and Y2 are each independently selected from the group consisting of -Cl, -Br, -NO2, -C = N, and -C = CH, and

the absolute stoichiometry at position 4 and 5 of the imidazoline ring is S and R, respectively.

Other compounds that are considered NTL analogs are compounds of formula 2 where R is piperazinyl substituted with at least one group selected from lower alkyl, cycloalkyl, -C = OR ⁱ , -N-lower alkyl, -SO2CH3, = 0, - CH2C = OCH3, ^or piperidinyl substituted with at least one group selected from C1-C3 alkyl, C1-C2 alkoxy, -C = 0CH3, -S02CH3'-C = 0, -OH, -CH2NH2, -C = 0CH2NH2, -C = 0CH20H, -C = 0CH (0H) CH20H, -CH2CH (0H) CH20H, -C = 0 N (CH2) 2, -C = 0 NH2, and -C = 0 N (CH3) CH3, -N (CH3) CH3, pyrrolidinyl and piperadinyl.

Also preferred are compounds of formula 2 wherein the group X I in the ortho position is selected from lower alkoxy, -OCH2CF3 and -OCH2CH2F, and the group X2 in the para position is lower alkoxy. Still more preferred are compounds wherein the group X I in the ortho position is selected from ethoxy, isopropoxy, -OCH2CF3 and -OCH2CH2F, and the group X2 in the para position is selected from methoxy and ethoxy.

More preferred are compounds of formula 2 wherein R is selected from piperazinyl and substituted piperazinyl. Such compounds are, for example: 1- {4 - [(4S, 5R) -4,5-Bis- (4-chloro-phenyl) -2- (2-isopropoxy-4-methoxy-phenyl) -4.5 -dihydro-imidazol-1carbonyl] -piperazin-1-yl} -ethanone; 4 - [(4S, 5R) -4,5-Bis- (4-chloro-phenyl) -2- (2-isopropoxy-4-methoxy-phenyl) -4,5-dihydroimidazole-1-carbonyl] piperazin-2-one ; [(4S, 5R) -4,5-Bis- (4-chloro-phenyl) -2- (2-isopropoxy-4-5-methoxy-phenyl) -4,5-dihydroimidazol-1-yl] - (4 -pyrrolidin-1-yl-piperidin-1-yl) -methanone; 4 - [(4S, 5R) -4,5-Bis- (4-chloro-phenyl) -2- (2-ethoxy-phenyl) -4,5-dihydro imidazole-1-carbonyl] -piperac ¡N-2one; 1- {4 - [(45.5R) -4,5-B¡s- (4-chloro-phen¡l) -2- (2-ethoxyphen¡l) -4,5-d¡h¡ dro-dazole-1-carbonyl] -piperazin-1-l} -ethanone; 2- {4 - [(45.5R) -4,5-B¡s- (4-doro-phen¡l) -2- (2-ethoxyphen¡l) -4,5-d¡h¡ dro-m¡dazoM-carbon¡l] -piperadn-1-¡l} -1-morpholin-4-¡l-ethanone; [(4S, 5R) -4,5-B¡s- (4-chloro-phen¡l) -2- (2-ethox¡-phen¡l) 4,5-d¡h¡dro-¡m¡ dazol-1-¡lH4- (2-methanesulfonylethyl) -piperadn-1-¡l] -methanone; and [(4S, 5R) -4,5-B¡s- (4-chloro-phen¡l) -2- (2-¡sopropox¡-4-methox¡-phen¡l) -4,5-dih ¡Dro-¡midazol-1-¡l] - (4-methylp¡peradn-1-yl) -methanone.

The synthesis of all of these molecules is known in the art, some of which are described in the '278 patent.

Peptides derived from the Cav-1 sequence

The caveolin-1 scaffold domain or peptide (Cav-1) (also referred to as CSD or CSP) interferes with the interaction of Cav-1 with Src kinases and mimics the combined effect of uPA and anti-p1-interggrin antibody as shown. discussed in more detail later. Native human Cav-1 is 178 amino acids long and has a molecular weight of 22 kDa. The amino acid sequence of Cav-1 is shown below (SEQ ID NO: 3).

1 M5GGKYVDSEGHLYTVPIREQGNIYKPNNK AMADELSEKQ VYDAHTKEID LVNRDPKHLN 61 DDWKIDFED VIAEPEGTHS FDGIWKASFT TFTVTKYWFY RLLSALFGIP MALIWGIYFAIE ILVCISIYFLTVVCISIYFA 121 ILVCISIHTVVKSWCDSIYFA ILSFLCIHVTVVKSWCDSIYFA IIFLCIHVTVVKSWCDSIYFA IIFFILCIHVKSVKSWCDSIYFA 121

CSP is the 20 residue peptide underlined above, and has the sequence GIWKASFTTFTVTKYWFYR (SEQ ID NO: 2). The preferred peptide of the present invention, designated CSP-4 is the FTTFTVT (SEQ ID NO: 1) fragment of CSP and is shown with double underlining in the Cav-1 sequence. CSP-4 has the activities shown in the examples and figures, below, and affects how p53 regulates LEC viability through coordinated control of uPA, uPAR (up) and PAI-1 (down) expression and by this presumed mechanism protects ECLs or lung epithelium in vivo from apoptosis and blocks fibrosis resulting from ALI.

In studies disclosed herein, a control peptide for CSP-4, which is called "CP" is a mixed peptide with the same amino acid composition as the major CSP (SEQ ID NO: 2), but has a different sequence: WGIDKAFFTTSTVTYKWFRY (SEQ ID NO: 5).

Modifications and changes can be made to the structure of CSP-4, and create molecules with similar or otherwise desirable characteristics. Such functional derivatives or biologically active derivatives (terms used interchangeably) are encompassed in the present disclosure.

Preferred functional derivatives are addition variants and peptide oligomers / multimers, and the like.

These can be generated synthetically, but also by recombinant production, and tested for the binding properties or biological activity of CSP-4. A preferred way to measure variant activity is in a competitive binding assay where the ability of the peptide variant to compete for soluble caveolin binding, such as one that is detectably labeled, with soluble uPAR ("suPAR" ).

It is understood that various derivatives of CSP-4 and longer polypeptides comprising CSP-4 can be readily made according to the invention, either by chemical (synthetic) methods or by recombinant means (preferred for longer polypeptides).

Included in the definition of functional variants of CSP-4 are addition which preferably comprises 1-5 additional amino acids at either end or both ends. In other embodiments (which are intended to be distinct from peptide multimers discussed below), more additional residues can be added, up to about 20 residues. In the CSP-4 addition variant, additional residues N-terminal to, and / or C-terminal to SEQ ID NO: 1 (the core CSP-4 peptide) may include some of those in the order in which they occur. in the native sequence in Cav-1 (SEQ ID NO: 4. However, the addition variant cannot be SEQ ID NO: 3. Alternatively, other amino acids can be added at either end of SEQ ID NO: 1, with the understanding that the spiked variant maintains the biological activity and binding activity of CSP-4 (at least 20% of the activity, or preferably more, as shown below).

Preferred substitution variants of CSP-4 are conservative substitutions in which 1, 2, or 3 residues have been replaced by a different residue. For a detailed description of protein chemistry and structure, see, Schultz GE et al., Principles of Prntein Structure, Springer-Verlag, New York, 1979, and Creighton, TE, Proteins: Structure and Molecular Properties, 2nd ed., WH Freeman & Co., San Francisco, 1993. Conservative substitutions and are defined herein as exchanges in one of the following groups:

Phe can be replaced by a large aromatic residue: Tyr, Trp.

Thr can be replaced by small, nonpolar or slightly polar aliphatic residues: eg Ala, Ser or Gly.

Val can be replaced by large, nonpolar aliphatic residues: Met, Leu, lie, Cys.

Even when it is difficult to predict the exact effect of a substitution before making it, one of skill in the art will appreciate that the effect can be evaluated by routine screening tests, preferably the biological and biochemical tests described herein. The activity of a purified cell lysate or polypeptide or peptide variant is screened in a suitable screening assay for the desired trait.

In addition to the 20 "standard" L-amino acids, D-amino acids or non-standard, modified or unusual amino acids that are well defined in the art are also contemplated for use in the present invention. These include, for example, include p-alanine (p-Ala) and other ^ -amino acids such as 3-aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so on; α-aminoisobutyric acid (Aib); £ -aminohexanoic acid (Aha); 6-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly); ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (Melle); phenylglycine (Phg); norleucine (Nle); 4-chlorophenylalanine (Phe (4-Cl)); 2-fluorophenylalanine (Phe (2-F)); 3-fluorophenylalanine (Phe (3-F)); 4-fluorophenylalanine (Phe (4-F)); penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,4-diaminobutyric acid (Dab); p-aminophenylalanine (Phe (pNH2)); N-methyl valine (MeVal); homocysteine (hCys), homophenylalanine (hPhe) and homoserine (hSer); hydroxyproline (Hyp), homoproline (hPro), N-methylated amino acids and peptoids (N-substituted glycines).

Other compounds can be engineered by rational drug design to function in a similar manner to CSP-4. The goal of rational drug design is to produce structural analogs of biologically active compounds. By creating such analogs, it is possible to produce drugs that are more active or more stable than natural molecules (ie, peptides), less susceptible to alterations that affect functions. One approach is to generate a three-dimensional structure of CSP-4, for example, by NMR or X-ray crystallography, computer modeling, or by a combination. An alternative approach is to randomly substitute functional groups in the CSP-4 sequence, and determine the effect on function.

Furthermore, a biologically active derivative has CSP-4 activity in an in vitro or in vivo binding or biological activity assay, such as the assays described herein. Preferably, the polypeptide inhibits or prevents apoptosis of BLM-induced SLE in vitro or in vivo with activity at least about 20% of CSP-4 activity, or at least about 30%, 40%, 50%, 60% , 65%, 70%, 75%, 80%, 85%, 90%, about 95%, 97%, 99%, and any differentiable range therein, such as, for example, from about 70% to about 80%, and more preferably from about 81% to about 90%; or even more preferably, from about 91% to about 99%. The derivative can have 100% or even higher activity than CSP-4.

The peptide may be protected at its N- or C-termini with an acyl group (abbreviated "Ac") and an amido (abbreviated "Am"), respectively, for example, acetyl (CH3CO-) at the N-terminus and amido (-NH2 ) at the C-terminus. A wide range of N-terminal protecting functions are contemplated, preferably at a linkage to the terminal amino group, eg, formyl;

alkanoyl, having 1 to 10 carbon atoms, such as acetyl, propionyl, butyryl;

alkenoyl, having 1 to 10 carbon atoms, such as hex-3-enoyl;

alkynoyl, having 1 to 10 carbon atoms, such as hex-5-ynoyl;

aroyl, such as benzoyl or 1 -naphthoyl;

heteroaroyl, such as 3-pyrroyl or 4-quinoloyl;

alkylsulfonyl, such as methanesulfonyl;

ariisulfonyl, such as benzenesulfonyl or sulfanilyl;

heteroarylsulfonyl, such as pyridine-4-sulfonyl;

substituted alkanoyl, having 1 to 10 carbon atoms, such as 4-aminobutyryl;

substituted alkenoyl, having 1 to 10 carbon atoms, such as 6-hydroxy-hex-3-enoyl;

substituted alkynoyl, having 1 to 10 carbon atoms, such as 3-hydroxy-hex-5-ynoyl;

substituted aroyl, such as 4-chlorobenzoyl or 8-hydroxy-naphth-2-oyl;

substituted heteroaroyl, such as 2,4-dioxo-1,2,3,4-tetrahydro-3-methyl-quinazolin-6-oyl;

substituted alkylsulfonyl, such as 2-aminoethanesulfonyl;

substituted ariisulfonyl, such as 5-dimethylamino-1-naphthalenesulfonyl;

substituted heteroarylsulfonyl, such as 1-methoxy-6-isoquinolininesulfonyl;

carbamoyl or thiocarbamoyl;

substituted carbamoyl (R'-NH-CO) or substituted thiocarbamoyl (R'-NH-CS) where R 'is alkyl, alkenyl, alkynyl, aryl, heteroaryl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted aryl, or heteroaryl replaced; substituted carbamoyl (R'-NH-CO) and substituted thiocarbamoyl (R'-NH-CS) where R 'is alkanoyl, alkenyl, alkynoyl, aroyl, heteroaroyl, substituted alkanoyl, substituted alkenoyl, substituted alkynoyl, substituted aroyl or substituted heteroaroyl , all as defined above.

The C-terminal protecting function can be an amide or ester bond with the terminal carboxyl. Protective functions that provide an amide bond are designated as NR1R2 where R1 and R2 can be independently extracted from the following group: hydrogen;

alkyl, preferably having 1 to 10 carbon atoms, such as methyl, ethyl, isopropyl;

alkenyl, preferably having 1 to 10 carbon atoms, such as prop-2-enyl;

alkynyl, preferably having 1 to 10 carbon atoms, such as prop-2-ynyl;

substituted alkyl having 1 to 10 carbon atoms, such as hydroxyalkyl, alkoxyalkyl, mercaptoalkyl, alkylthioalkyl, haloalkyl, cyanoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkanoylalkyl, carboxyalkyl, carbamoylalkyl;

Substituted alkenyl having 1 to 10 carbon atoms, such as hydroxyalkenyl, alkoxyalkenyl, mercaptoalkenyl, alkylthioalkenyl, haloalkenyl, cyanoalkenyl, aminoalkenyl, alkylaminoalkenyl, dialkylaminoalkenyl, alkanoylalkenyl, carboxyalkenyl, alkanoylalkenyl, carboxyalkenyl;

substituted alkynyl having 1 to 10 carbon atoms, such as hydroxyalkynyl, alkoxyalkynyl, mercaptoalkynyl, alkylthioalkynyl, haloalkynyl, cyanoalkynyl, aminoalkynyl, alkylaminoalkynyl, dialkylaminoalkynyl, alkanoylalkynyl, carboxyalkynyl, carbamoylalkynyl;

aroylalkyl having up to 10 carbon atoms, such as phenacyl or 2-benzoylethyl;

aryl, such as phenyl or 1-naphthyl;

heteroaryl, such as 4-quinolyl;

alkanoyl having 1 to 10 carbon atoms, such as acetyl or butyryl;

aroyl, such as benzoyl;

heteroaroyl, such as 3-quinoloyl;

OR 'or NR'R "where R' and R" are independently hydrogen, alkyl, aryl, heteroaryl, acyl, aroyl, sulfonoyl, sulfinyl, ^or SO2-R "' ^or SO-R"' where R "'is alkyl, substituted ^or unsubstituted aryl, heteroaryl, alkenyl, ^{or alkynyl.}

Protective functions that provide an ester bond are designated as OR, where R can be: alkoxy; aryloxy; heteroaryloxy; aralkyloxy; heteroaralkyloxy; substituted alkoxy; substituted aryloxy; substituted heteroaryloxy; substituted aralkyloxy; or substituted heteroaralkyloxy.

The N-terminal or C-terminal protective functions, or both, can be of such a structure that the protected molecule functions as a prodrug (a pharmacologically inactive derivative of the parent drug molecule) that undergoes spontaneous or enzymatic transformation in the body with in order to release the active drug and having improved delivery properties on the parent drug molecule (Bundgaard H, Ed: Design of Prodrugs, Elsevier, Amsterdam, 1985).

The judicious choice of protecting groups allows the addition of other activities on the peptide. For example, the presence of a sulfhydryl group attached to the N- or C-terminal protection will allow conjugation of the derivatized peptide to other molecules.

Chemical derivatives of CSP-4

In addition to the protecting groups described above that are considered "chemical derivatives" of CSP-4, preferred chemical derivatives of CSP-4 may contain additional chemical moieties not normally a part of a protein or peptide that can be introduced into CSP-4 or a variant of addition of CSP-4 by known means to constitute the chemical derivative as defined herein. Covalent modifications of peptides are included within the scope of this invention. Such derivatized fractions can improve solubility, absorption, biological half-life, and the like. Fractions capable of mediating such effects are disclosed, for example, in Gennaro, AR, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins Publishers; 21st Ed, 2005 (or latest edition).

Such modifications can be introduced into the molecule by reacting target amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Another modification is peptide cyclization - which is generally accomplished by adding terminal Cys residues that can be attached via a disulfide bond to generate a cyclic peptide. Alternatively, a cross-linkable Lys (K) is added at one end and a GIu (E) at the other end.

Cysteinyl residues (added, eg, for cyclization purposes) are most commonly reacted with α-haloacetates (and corresponding amines) to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-p- (5-imidozoyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyl disulfide , p-chloromercuribenzoate, 2-chloromercuri-4-nitro-phenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

The added lysinyl residues (eg, for cyclization) and the amino terminal residue can be derivatized with succinic acid anhydrides or other carboxylic acids. Derivatization with a cyclic carboxylic anhydride has the effect of reversing the charge on lysinyl residues. Other suitable reagents for derivatizing amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentaodione; and transaminase catalyzed reaction with glyoxylate.

Derivatization with bifunctional agents is useful for crosslinking the peptide or oligomer or multimer to a water insoluble support matrix or other macromolecular support. Commonly used crosslinking agents include 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, 4-azidosalicyclic acid esters, homobifunctional imidoasters, including disuccinimidyl esters such as 3,3'-dithiob S (sucdnimidl propionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3 - [(p-azidophenil) ditio] propimidate give photoactivatable intermediates that are capable of crosslinking in the presence of light. Alternatively, reactive water insoluble matrices such as cyanogen bromide activated carbohydrates and reactive substrates described in US Patents 3,969,287, 3,691,016, 4,195,128, 4,247,642, 4,229,537, and 4.330.44o are used for protein immobilization.

Other modifications include hydroxylation of lysine, phosphorylation of the hydroxyl groups of threonyl residues, methylation of the a-amino groups of the side chains of the chains of lysine residues (added) (Creighton, supra), acetylation of the amine N- terminal and, in some cases, amidation of the C-terminal carboxyl groups. Also included are peptides where one or more D-amino acids are replaced by one or more L-amino acids.

Multimaric or oligomaric peptides

The present disclosure also includes longer peptides constructed from CSP-4 repeating units or a functional derivative thereof that has the anti-apoptotic and protective activity of CSP-4. The peptide unit of such a multimer is FTTFTVT (SEQ Id NO: 1). Addition variants of this peptide that can be the "unit" of the multimer preferably include from 1-4 additional amino acids.

A peptide multimer can comprise different combinations of peptide monomers (which can include one or both of SEQ ID NO: 1 or addition variants thereof or a chemically derivatized form of the peptide. Such oligomaric or multimaric peptides can be made by chemical synthesis or by recombinant DNA techniques as discussed herein. When produced by chemical synthesis, oligomers preferably have 2-5 repeats of a core peptide sequence, and the total number of amino acids in the multimer should not exceed about 160 residues, preferably no more than 100 residues (or their equivalents, when they include linkers or spacers).

A preferred synthetic chemical peptide multimer has the formula

P1n

wherein the core peptide P1 is SEQ ID NO: 1, and where n = 2-5, and where the core peptide alone or in oligo- or multimalar form has the biological activity of Cs P-4 as disclosed in the present document in an in vitro or in vivo bioassay of such activity.

In another embodiment, a preferred synthetic chemical peptide multimer has the formula

(P1-X ^m ) ⁿ -P2

P1 and P2 are the core peptides described above, including additional variants, where

(a) P1 and P2 can be the same ^or different; furthermore, each occurrence of P1 in the multimer may be a different (or variant) peptide

(b) X is a spacer comprising or consisting of

(i) a short organic chain, preferably C1-C5 alkyl, C1-C5 alkenyl, C1-C5 alkynyl, C1-C5 polyether containing up to 4 oxygen atoms, where m = 0 ^or 1 and n = 1 -7; ^or

(¡I) Glyz where z = 1-6,

and wherein the core peptide alone or in oligo- or multimalar form has the biological activity of CSP-4 as disclosed herein in an in vitro or in vivo bioassay of such activity.

When produced recombinantly, a preferred spacer is Glyz as described above, where z = 1-6, and multimers can have as many repeats of the core peptide sequence as the expression system allows, for example, from two to about 25 repetitions. A preferred recombinantly produced peptide multimer has the formula:

(P1-Glyz) n-P2

where

(a) P1 and P2 are, independently, SEQ ID NO: 1 or 3 or an addition variant or derivatized form thereof, wherein P1 and P2 may be the same or different; furthermore, each occurrence of P1 in the multimer may be a different (or variant) peptide

where

n = 1-25 and z = 0-6; ( Preferred ranges of n include n = 1-5, 1-10, 1-15, or 1-20) and wherein the core peptide alone or in multimeric form has the biological activity of CSP-4 as disclosed herein. document in an in vitro or in vivo bioassay of such activity.

Ios present peptide, P1 or P2 multimers is preferably SEQ ID NO: 1 or a variant or chemical derivative of adding the same. The multimer is optionally protected. It is understood that such multimers may be built from any of Ios peptides or variants described herein. It is also understood that the peptide multimer must be different from SEQ ID NO: 3 (ie, not native human Cav-1 and is preferably not a native mammalian Cav-1 homolog).

Peptidomimetics

A peptidomimetic compound that mimics biological effects Ios CSP-4 is also disclosed. A peptidomimetic agent may be a non - natural peptide or non - peptide agent that recreates estereoespaciales properties Ios connecting elements 4 so-CSP having binding activity and biological activity of CSP-4. Similar to a biologically active CSP-4 peptide, peptide multimer, a peptidomimetic will have a binding face (which interacts with any ligand to which CSP-4 binds) and a non-binding face. Again, similar to CSP-4, the non-binding side of a peptidomimetic will contain functional groups that can be modified by coupling various therapeutic moieties without modifying the binding side of the peptidomimetic. A preferred embodiment of a peptidomimetic would contain an aniline on the non-binding side of the molecule. The NH2 group of an aniline has a pKa ~ 4.5 and could therefore be modified by any NH2 selective reagent without modifying any NH2 functional group on the peptidomimetic bonding face. Other peptidomimetics may have no NH2 functional group on their binding side and thus any NH2, regardless of pKa could show up on the non-binding side as a site for conjugation. In addition, other modifiable functional groups, such as -SH and -COOH could be incorporated into the non-binding side of a peptidomimetic as a conjugation site. A therapeutic moiety could also be directly incorporated during the synthesis of a peptidomimetic and preferably displayed on the non-binding side of the molecule.

Compounds that retain partial peptide characteristics are further disclosed. For example, any proteolytically unstable bond in a peptide of the invention could be selectively substituted for a non-peptidic element such as an isosterone (N-methylation; D-amino acid) or a reduced peptide bond while the rest of the molecule reflects its nature. peptide.

Peptidomimetic compounds, whether agonists, substrates or inhibitors, have been described for a number of bioactive peptides / polypeptides such as opioid peptides, VIP, thrombin, HIV protease, etc. Methods for designing and preparing peptidomimetic compounds are known in the art (Hruby, VJ, Biopolymers 33: 1073-1082 (1993); Wiley, RA et to the, Med Res Rev. 13:... 327-384 (1993); Moore et al., Adv. In Pharmacol 33: 91-141 (1995); Giannis et al., Adv. In Drug Res. 29: 1-78 (1997) Certain mimetics that mimic secondary structure are described in Johnson et al. al., In: Biotechnology and Pharmacy, Pezzuto et al., Chapman and Hall (Eds.), NY, 1993. These methods are used to make peptidomimetics that possess at least the binding capacity and specificity of the CSP-4 peptide and preferably also possess biological activity. knowledge of peptide chemistry and organic chemistry generally available to experts in the art Ios is sufficient, in view of this disclosure, to design and synthesize such compounds.

For example, such peptidomimetics have been identified by inspection of the three-dimensional structure of a free or complexed peptide of the invention with a ligand (eg, soluble uPAR or a fragment thereof). Alternatively, the structure of a peptide of the invention bound to its ligand can be obtained by nuclear magnetic resonance spectroscopy techniques. A better understanding of the stereochemistry of the interaction of the peptide with its ligand or receptor will allow the rational design of such peptidomimetic agents. The structure of a peptide or polypeptide of the invention in the absence of ligand could also provide a scaffold for the design of mimetic molecules.

Administered peptides

One embodiment of the disclosure comprises a method of introducing the peptide of the invention into animal cells, such as human cells. Compositions useful for this method, called "administrable" or "administrable to cells" or "cell-targeted" peptides or polypeptides comprise a biologically active peptide according to the invention, preferably CSP-4, or a functional derivative thereof, or a multimer. peptide thereof, having attached thereto or associated with, an additional component that serves as an "internalizing sequence" or cellular delivery system. The term "associated with" can include chemically bonded or coupled to, either by covalent or other bonding or forces, or combined with, as in a mixture. As used herein, "delivery" refers to internalizing a peptide / polypeptide into a cell. Delivery molecules contemplated herein include peptides / polypeptides used by others to effect cellular entry. See, for example, Morris et al., Nature Biotechnology, 19: 1173-6, 2001). A preferred strategy is as follows: an apoptosis inhibitor ("biologically active") peptide of the invention is bound to or mixed with a specially designed peptide that facilitates its entry into cells, preferably human cells. This system of Administration does not require that the delivery peptide be fused or chemically coupled to the biologically active peptide or polypeptide (although preferred), nor does the biologically active peptide or polypeptide have to be denatured prior to the process of administration or internalization. A disadvantage of prior delivery systems is the requirement for denaturation of the "cargo" protein prior to administration and subsequent intracellular restoration. These embodiments are based on known approaches to promote protein translocation to cells.

One type of "delivery" peptide / polypeptide that promotes translocation / internalization includes the HIV TAT protein (Frankel, AD et al., Cell 55: 1189-93 (1998), and the third a-helix of the Antennapedia homeodomain ( Derossi et al., J. Biol. Chem. 269: 10444-50 (1994); Lindgren, M et al., Trends Pharm. Scí. 21: 99-103 (2000); Lindgren et al., Bioconjug Chem. ( 2000); Maniti O et al., PLoS ONE 5e15819 (2010). The latter peptide, also known as "penetratin" is a 16 amino acid peptide with the wild-type sequence Rq IKIWFQNRRMKWKK (SEQ ID NO: 6) or two analogs / variants designated W48F (RQIKIFFQNRRMKWKK, SEQ ID NO: 7) and W56F (RQIKIWFQNRRMKFKK, SEQ ID NO: 8) (Christiaens B et al., Eur Biochem 2002, 269: 2918-2926). Another variant with both of the above mutations en RQIKIFFQNRRMKFKK (SEQ ID NO: 9). Transportan, a cell-penetrating peptide is a 27 amino acid long peptide containing 12 functional amino acids from the N-terminus of the neuropeptide galanin linked by a Lys residue added to the mastoparan sequence (Pooga, M et al., FASEB J.

12: 67-77 (1998)). The transportan sequence is GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 10). Penetratin and transportan analogs are described by Lindgren et al., Bioconjug Chem. 2000.

Another (family of) protein includes VP22, a herpes simplex virus protein that has the remarkable property of intracellular transport and distributes a protein to many surrounding cells (Elliott, G et al., 1997, Cell 88: 223-33; O 'Hare et al., US Patent 6,017,735). For example, VP22 bound to p53 (Phelan, A. et al., 1998, Nat Biotechnol 16: 440-3) or thymidine kinase (Dilber, MS et al., 1999, Gene Then 6: 12-21) facilitates propagation of proteins bound to surrounding cells in vitro. Also useful are homologs of VP22 in other herpesviruses, such as the avian Marek's disease virus (MDV) protein UL49, which shares homology with VP22 of HSV-1 (Koptidesova et al., 1995, Arch Virol. 140 : 355 -62) and has been shown to be capable of intracellular transport after exogenous application (Dorange et al., 2000, J Gen Virol. 81: 2219). All of these proteins share the property of intercellular propagation that provides an approach to increasing cellular uptake of peptides, variants, and multimers of this invention.

Also included are "functional derivatives" of the above intracellularly propagating or "delivery" proteins and peptides "delivery" or "internalization" such as HIV TAT or VP22 that include homologous amino acid substitution variants, chemical fragments or derivatives, terms that they are herein for biologically active peptides. A functional derivative retains quantifiable intercellular translocation or propagation activity (VP22 type) that encourages entry of the desired polypeptide, which promotes the utility of the present biologically active peptide, eg, for therapy. "Functional derivatives" encompass variants (preferably conservative substitution variants) and fragments regardless of whether the terms are used in linker or alternative.

Since the above transport proteins are said to work best when conjugated or otherwise bound to the peptide they transport, such as CSP-4 or a variant or multimer thereof, there are a number of disadvantages to using them. A more efficient delivery polypeptide that can be mixed with the biologically active peptide and does not need to be chemically linked for action is described in Morris et al., Supra, as "Pep-1" having the amphipathic amino acid sequence KeTWWETw Wt EWSQPKKKRKV (SEQ ID NO: 11). Pep -1 consists of three domains:

(1) a Trp-rich hydrophobic motif containing five KETWWETWWTEW Trp residues (residues 1-12 of SEQ ID NO: 11, above). This motif is desirable, or required, to effectively target the cell membrane and to enter into hydrophobic interactions with proteins;

(2) a hydrophilic domain rich in Lys KKKRKV (the 6 C-terminal residues of SEQ ID NO: 11) which is derived from the nuclear localization sequence of the large T antigen of the SV40 virus, and improves intracellular delivery and solubility of the peptide; and

(3) an SQP spacer "domain" (3 internal residues of SEQ ID NO: 12) that and separates the two previous active domains and includes a Pro that improves the flexibility and integrity of both hydrophobic and hydrophilic domains.

Accordingly, another embodiment of the disclosure is an administrable peptide or polypeptide comprising CSP-4 or a functional derivative thereof as described above, and a molecule or delivery or translocation fraction bound thereto or associated therewith. . The delivery molecule can be a peptide or polypeptide, for example,

(a) HIV TAT protein or a translocationally active derivative thereof,

(b) penetratin having the sequence Rq IKIWFQNRRMKWKK (SEQ ID NO: 8),

(c) a W48F penetratin variant having the sequence RQIKIFFQNRRMKWKK (SEQ ID NO: 7)

(d) a W56F penetratin variant having the sequence RQIKIWFQNRRMKFKK (s EQ ID NO: 13)

(e) a penetratin variant having the sequence RQIKIWFQNRRMKFKK (SEQ ID NO: 16) (f) transportan having the sequence GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 10)

(g) VP22 protein from herpes simplex virus ^or a translocationally active homolog thereof from a different herpesvirus such as MDV UL49 protein; or

(h) Pep-1 having the sequence KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 9).

When a delivery moiety, such as the peptides and proteins discussed above, is conjugated or fused to the biologically active peptide of the invention, it is preferred that the delivery moiety is N-terminal to the biologically active peptide.

In vitro assay of compositions

The compounds of this invention are tested for their biological activity, eg, antifibrotic activity, their ability to affect the expression of uPA, uPAR and PAI-1 mRNAs, inhibit the proliferation of lung fibroblasts, etc., using any of the assays described and / or exemplified herein or others well known in the art.

In vivo testing of compositions

The ability of a compound (such as NTL analogs or variants or derivatives or peptide multimers of CSP-4 to inhibit pulmonary fibrosis in BLM-treated mice is a preferred assay to assess the functional activity of the compound. Other known assays can also be used in technique that measure the same type of activity.

Method of preventing or treating lung injury or fibrosis

The compounds and compositions described herein are used in a method for inhibiting the interaction of MDM2 with the p53 protein in vitro or in vivo, and for treating diseases or conditions associated with ALI and pulmonary fibrosis / IPF.

Pharmaceutical and therapeutic compositions and their administration

Compounds that can be employed in the pharmaceutical compositions of the disclosure include NTL, and all of the peptide compounds described above, as well as the pharmaceutically acceptable salts of these compounds. "Pharmaceutically acceptable salt" refers to conventional acid addition salts or base addition salts that retain the biological efficacy and properties of the compounds of the present invention and are formed from suitable non-toxic inorganic or organic acids or inorganic or organic bases. Sample acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, acid salicylic, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Sample base addition salts include those derived from ammonium, potassium, sodium and quaternary ammonium hydroxides, such as, for example, tetramethylammonium hydroxide. Chemical modification of a pharmaceutical compound (ie, drug) to a salt is a well-known technique for pharmaceutical chemists to obtain improved physical and chemical stability, hygroscopicity, fluidity, and solubility of compounds. See, for example, H. Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 196 and 1456-1457.

As stated above, the compounds of the invention possess the ability to inhibit the interaction of MDM2 with the p53 protein and are used in the treatment of acute lung injury and, in particular, pulmonary fibrosis.

The compounds of the invention, as well as the pharmaceutically acceptable salts thereof, can be incorporated into convenient dosage forms, such as capsules, impregnated wafers, tablets, or preferably, injectable preparations. Solid or liquid pharmaceutically acceptable carriers can be used. "Pharmaceutically acceptable", such as a carrier, excipient, etc. "pharmaceutically acceptable" means pharmacologically acceptable and substantially non-toxic to the subject to whom the particular compound is administered.

Solid supports include starch, lactose, calcium sulfate dihydrate, white clay, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Liquid carriers include syrup, peanut oil, olive oil, saline, water, dextrose, glycerol, and the like. Similarly, the carrier or diluent can include any sustained release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax. When a liquid carrier is used, the preparation may be in the form of a syrup, an elixir, an emulsion, a soft gelatin capsule, a sterile injectable liquid (for example, a solution), such as an ampoule, or an aqueous or non-aqueous liquid suspension. watery. A summary of such pharmaceutical compositions can be found, for example, in Gennaro, AR, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins Publishers; 2 ia Ed, 2005 ( ^or latest edition).

Pharmaceutical preparations are made according to conventional techniques of pharmaceutical chemistry involving steps such as mixing, granulation and compression, when necessary for tablet forms, or mixing, filling and dissolving the ingredients, as appropriate, to give the desired products for administration. oral, parenteral, topical, transdermal, intravaginal, intrapenile, intranasal, intrabronchial, intracranial, intraocular, intra-atrial and rectal. The pharmaceutical compositions may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH regulating agents, and so on.

The present invention can be used in the treatment of any of a number of animal genera and species, and are equally applicable in the practice of human or veterinary medicine. Thus, the pharmaceutical compositions can be used to treat domestic and commercial animals, including birds and more preferably mammals, most preferably humans.

The term "systemic administration" refers to administration of a composition or agent such as NTL or the peptides described herein, in a way that causes introduction of the composition into the subject's circulatory system or otherwise allows its spread. throughout the body, such as intravenous (IV) injection or infusion. "Regional" administration refers to administration to a specific, and somewhat more limited, anatomical space, such as instillation into the lung, the preferred route, or intrapleural, intraperitoneal, intrathecal, subdural, or to a specific organ. Other examples include intranasal, which is a route that corresponds to instillation into the lungs, intrabronchial, intra-atrial, or intraocular, etc. The term "local administration" refers to the administration of a composition or drug in a limited, or circumscribed anatomical space, such as subcutaneous injections (s.c.), intramuscular injections (i.m.). One skilled in the art would understand that the local administration or the regional administration often also produce input of a composition into the circulatory system, that is, so that s.c. or i.m. they can also be routes for systemic administration. Instilable, injectable or infusible preparations can be prepared in conventional forms, either as solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection or infusion, or as emulsions. Although the preferred regional routes of administration are to the lungs, the pharmaceutical composition can be administered systemically or topically or transdermally either separately from, or concurrently with, instillation into the lungs.

Other pharmaceutically acceptable carriers for compositions of the present invention are liposomes, pharmaceutical compositions in which the active polypeptide is contained either dispersed or variously present in corpuscles consisting of concentric aqueous layers adherent to lipid layers. The active polypeptide is preferably present in the aqueous layer and in the lipid layer, inside or outside, or, in any case, in the generally non-homogeneous system known as a liposomal suspension. The hydrophobic layer, or lipid layer, in general, but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surfactants such as dicetyl phosphate, stearylamine or phosphatidic acid, and / or other materials of a hydrophobic nature. Those skilled in the art will appreciate other suitable embodiments of the present liposomal formulations.

The therapeutic dose administered is an amount that is therapeutically effective, as is known or is readily determinable by those skilled in the art. The dose also depends on the age, health, and weight of the recipient, type of concurrent treatment (s), if any, the frequency of treatment, and the nature of the effect desired.

Therapeutic methods

The methods of this disclosure can be used to treat pulmonary fibrosis or IPF in a subject in need thereof. The term "treat" is broadly defined to include at least the following: inhibit, reduce, ameliorate, prevent, reduce the occurrence or relapse, including the frequency and / or time to relapse, or the severity of the symptoms of the disease or condition being treated or prevented. This can occur as a result of inhibiting epithelial cell death, inhibiting fibroblast proliferation, any of the other biological or biochemical mechanisms disclosed herein that are associated with or responsible for IPF.

The NTL, NTL analog, or peptide or peptide derivative or pharmaceutically acceptable salt thereof is preferably administered in the form of a pharmaceutical composition as described above.

Doses of the compound preferably include pharmaceutical dosage forms comprising an effective amount of the NTL or peptide. The dosage form refers to physically discrete units suitable as unit doses for a mammalian subject; each unit contains a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. The specification of the pharmaceutical forms of the invention is dictated by and depends directly on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technique of composition of such active compound for the treatment of, and sensitivity of, individual subjects.

By an effective amount is meant an amount sufficient to achieve a regional concentration or steady state concentration in vivo that produces a measurable reduction in any relevant disease parameter.

The amount of active compound to be administered depends on the selected NTL, NTL analog, peptide or derivative thereof, the precise disease or condition, the route of administration, the health and weight of the recipient, the existence of other concurrent treatment. , if any, the frequency of treatment, the nature of the effect desired, and the expert judgment.

A preferred individual dose, given once daily to treat a subject, preferably a mammal, more preferably a human suffering from or susceptible to IPF resulting therefrom is between about 0.2 mg / kg and about 250 mg / kg. , preferably it is between about 10 mg / kg and about 50 mg / kg, for example, by instillation (by inhalation). Such a dose can be administered daily from anywhere from about 3 days to one or more weeks. Chronic administration is also possible, although it may be necessary to adjust the dose downward as is well understood in the art. The above ranges are, however, indicative, as the number of variables in an individual treatment regimen is large, and considerable shifts from these preferred values are expected.

For continuous administration, for example by a pump system such as an osmotic pump that was used in some of the experiments described below, a total dose over a time of about 1-2 weeks is preferably in the range of 1 mg / kg to 1 g / kg, preferably 20-300 mg / kg, more preferably 50-200 mg / kg. After such continuous dose regimen, the total concentration of the active compound is preferably in the range of about 0.5 to about 50 | j M, preferably about 1 to about 10 | j M.

An effective concentration of the active compound to inhibit or prevent inhibiting apoptosis in vitro is in the range of about 0.5 nM to about 100 nM, more preferably about 2 nM to about 20 nM. Optimal effective doses and dose ranges can be determined in vitro using the methods described herein.

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Expression of p53 in FL fibroblasts decreased in fibrotic foci of IPF lungs

Staining of lung sections from IPF patients and "normal" control subjects with various reagents, including immunohistochemistry (IHC) (see, Figures 2A-2I) revealed that IPF tissues show fibrotic foci that are dense with ECM. The FL fibroblasts dispersed in the vimentin-rich foci showed minimal staining for p53 and PAI-1 antigens. However, immunofluorescence staining for SP-C, PAI-1, p53, and active caspase-3 (not shown) demonstrated that type II alveolar cells (ATII) surrounding fibrotic foci show elevated staining for p53 and Pa I antigens. -1, and are positive for active caspase-3, indicating apoptosis of surrounding ATII cells. The iHc results reveal that the ATII cells that enclose the fibrotic foci continually die due to increased expression of p53 and PAI-1. These wounds are replaced by activated fibroblasts showing minimal basal p53 and PAI-1 staining, and elevated Ki-67 staining, indicating proliferation due to suppression of p53 and PAI-1 expression.

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Caveolin-1, p53 and PAI-1 decreased and uPA increased in human FL fibroblasts from IPF lungs

NL and FL fibroblast cell lysates were immunoblotted to reveal changes in proteins and miRNAs. See Figures 3A-3B. Baseline miR-34a expression was significantly lower in FL fibroblasts indicating that reduced p53 expression and consequent changes in the p53-uPA fibrinolytic system interface contributed to fibrogenesis. Such changes are associated with increased chol-I and inhibition of miR-34a. The results further showed that miR-34a-mediated increases in miR-34a transcription induced by p53 or stabilization of p53 in human FL fibroblasts can mitigate pulmonary fibrosis.

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Uneven expression of uPA, PAI-1, and col-I mRNAs by FPI and "normal" lung fibroblasts

Total RNA isolated from lung tissues of "normal" subjects and IPF or NL patients was assayed for uPA, PAI-1 and col-I mRNA by quantitative RT-PCR and normalized to corresponding levels of p-actin mRNA. . The results are shown in Figures 4A-4B.

The expression of col-I and PAI-1 mRNA was significantly increased in FPI lung tissues while uPA mRNA was decreased. Interestingly, unlike FPI lung tissues, the mRNA of col-I and uPA and lower level of PAI-1 mRNA expression was found in FL fibroblasts compared to NL fibroblasts. UPAR protein and mRNA are also elevated in FL fibroblasts. Elevated PAI-1 in lung tissues is attributable to increased expression of PAI-1 by lung epithelial cells or macrophages rather than FL fibroblasts (IPF). This is consistent with the increased expression of coI-I, uPA and uPAR proteins and reduced PAI-1 in FPI lung FL fibroblasts (see Figure 3A).

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Differential expression of uPA and PAI-1 by FL fibroblasts versus NL from mice

FL fibroblasts were isolated from mouse lungs 21 days after BLM injury or NL fibroblasts from control mice exposed to intranasal saline as described elsewhere26. Fibroblast lysates NL and FL were immunoblotted to

Changes in the expression of p53, uPA, PAI-1 and coI-I proteins were detected by immunoblotting of NL and FL fibroblast cell lysates (Figs. 5A-5D). Total RNA was extracted from mouse lung tissues with NL and FL fibroblasts from lung tissues of BLM-induced or control (saline-treated) mice with mRNA and assayed for uPA, PAI-1 and CoI-I mRNA. The proliferative rates of mouse NL and f L fibroblasts were compared. Consistent with FL fibroblasts (FPI) from FPI2 lungs, the basal proliferation rate of murine Fl fibroblasts (BLM) was significantly higher than that of NL fibroblasts (from saline-treated mice). p53 inhibited uPA gene transcription, at the same time bound to the PAI-1 promoter and increased transcription of PAI-133-34 mRNA p53 also inhibited uPA and uPAR expression by destabilizing their transcripts while stabilizing the PAI-123'35 transcript. Finally, the inhibition of uPA or uPAR increases the expression of p53 and subsequent induction of PAI-1 mediated by p53, thus increasing cell senescence and apoptosis. It was concluded that p53-mediated regulation of the uPA fibrinolytic system at transcriptional and post-transcriptional levels is compromised in FL fibroblasts due to lack of p53 expression. This leads to increased uPA and uPAR and inhibition of PAI-1 in these cells.

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CSP increases p53 expression through induction of miR-34a in FL fibroblasts (IPF)

Fibroblasts NL and FL were treated with PBS, CSP or CP and RNA for miR-34a was analyzed by real-time PCR. Furthermore, FL fibroblasts were cultured in the presence of PBS or CSP or CP with or without antisense miR-34a (miR-34a AS) or pre-miR-34a (or control miRNA. Conditioned medium (CM) of these cultures was immunoblotted for PAI -1 and cell lysates (CL) were immunoblotted for p53. In one experiment, FL fibroblasts were treated with PBS or Cs P or CP in the presence or absence of miR-34a AS, pre-miR-34a or control miR. RNA for changes in miR-34a by real-time PCR Results appear in Figures 6A-6C.

A series of overlapping deletions were made in CSP and these peptide fragments were assayed in FL fibroblasts for changes in p53, uPA, PAI-1 and coI-I (Fig. 6D).

CSP significantly increased miR-34a and p53 expression in FL fibroblasts, but not NL. Induction of p53 by CSP was mimicked by expression of pre-miR-34a alone and abolished by inhibition of miR-34a by miR-34a AS. This indicated that the induction of p53 occurs by miR-34a mediated stabilization.

Deletion analysis of the 20mer CSP revealed that an internal 7 amino acid fragment, called CSP-4 and having the sequence FTTFTVT (SEQ ID NO: 1) had the full length peptide activity. This supports the conclusion that the beneficial effect of CSP-4 in FL fibroblasts or mice with established pulmonary fibrosis (see Figures 11A-11D), involves control of p53 and miR-34a expression.

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Increased mdm2-mediated degradation of p53 proteins in FL fibroblasts (IPF)

Fibroblast lysates NL and FL were immunoblotted for p53 and mdm2, or immunoprecipitated with anti-imdm2 antibody and immunoblotted (IB) for the associated p53 protein. The results showed robust mdm2-p53 interaction in NL fibroblasts that were absent in FL fibroblasts (see Figures 7A-7B) despite elevated levels of mdm2 in FL fibroblasts. Thus, the increased mdm2-mediated degradation of the p53 protein contributed to the marked suppression of basal p53 levels in FL fibroblasts, supporting the use of the therapeutic interventions disclosed herein to target the interaction of mdm2 and p53.

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Nutlin-3a (NTL) or CSP-4-mediated inhibition of coI-I expression in FL fibroblasts (FPI): Role of the p53-fibrinolytic system interrelation

FL fibroblasts were exposed to PBS (control), nutlin-3a (10 mM), CSP-4 (10 nM) or CP for 48 hours. Lysates were immunoblotted for the expression of CoI-I, PAI-1, p53 and uPA. The results are shown in Figures 8A-8B. Cultured FLs were exposed to vehicle (PBS), ^or nutlin-3a, CSP-4 ^or CP and counted to assess proliferation. The results showed that both nutlin-3a and CSP-4 inhibited the proliferation of FL fibroblasts. The process involves induction of the expression of p53 and PAI-1 and concurrent inhibition of uPA.

In another experiment (results in Figure 8C), FL fibroblasts were transduced with adenovirus vector expressing p53, PAI-1, or caveolin-1 or appropriate vector controls and cultured for 2 days when the cell lysates were immunoblotted. for CoI-I, p53, PAI-1 and uPA. Transduction of FPI lung FL fibroblasts with Ad-Cav-1 induced p53 and PAI-1 while inhibiting the expression of uPA and coI-I. The effects of nutlin-3a or CSP-4 on FL fibroblasts were mimicked by expressing p53 or PAI-1 or caveolin-1 alone. Thus, p53-mediated induction of PAI-1 and inhibition of uPA expression contribute to the inhibition of coI-I. Interestingly, treatment of FL fibroblasts from the lungs of IPF patients or BLM-treated mice with recombinant PAI-1 protein failed to induce apoptosis or senescence (not shown) while exogenous elevated PAI-1 induced apoptosis of both ATII cells. in vitro and in vivo. The resistance of f L fibroblasts to exogenous PAI-1 would allow these cells to thrive in media rich in PAI-1 proteins from IPF or BLM-damaged lungs.

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Role of the p53-fibrinolytic system interrelation in the induction of coI-I expression in NL fibroblasts

Histologically "normal" human lung NL fibroblasts 2 were transfected with a lentivirus vector bearing p53 shRNA to inhibit basal p53 expression. Control cells were similarly treated with a non-specific shRNA. Culture-conditioned medium was immunoblotted for PAI-1, uPA, and soluble coI-I, and cell lysates were assayed for p53 and ci-SMA. Total RNA isolated from these fibroblasts was analyzed for changes in mRNA expression of uPA, pA | -1 and coI-I by quantitative RT-PCR. The results (in Figures 9A-9B) show that inhibition of p53 expression in NL fibroblasts increased uPA, coI-I and ci-SMA, but inhibited PAI-1. This supports the link described here between p53-mediated changes in the uPa system and fibrogenesis.

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Nutlin-3a inhibits pulmonary fibrosis in BLM-treated mice

Mice exposed to BLM (or saline in controls) for 14 d to induce pulmonary fibrosis were injected IV with nutlin-3a (10 mg / kg body weight) 35 ^or a vehicle control to determine and the effect on established pulmonary fibrosis was examined. . Fibrosis was assessed by CT and lung function (compliance and resistance) were measured using the Flexivent system. Pulmonary sections were subjected to trichrome staining and H&E staining (the latter not shown) to assess lung architecture and collagen deposition as an indication of fibrosis. Finally, whole lung homogenates were analyzed for total collagen (hydroxyproline) and desmosine26,36 content as an independent evaluation of changes in ECM. The results (Fig. 10-10D) of all the previous trials indicated a beneficial effect of nutlin-3a in pulmonary fibrosis. Oral administration of nutlin-3a 14 days after BLM injury also inhibited pulmonary fibrosis in mice.

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CSP-4 peptide inhibits established lung fibrosis and improves lung function

Mice were challenged with BLM to induce pulmonary fibrosis. After 14 d, the mice were injected IV with vehicle or 1.5 mg / kg of body weight of

CSP-4 (SEQ ID NO: 1) or control peptide (mixed sequence of the same amino acids; CP, SEQ ID NO: 5)) at 1.5 mg / kg body weight or vehicle was injected IV into BLM-challenged mice 14 days before. One week later, the mice were tested by CT for pulmonary fibrosis. Lung volume was measured in the same mice using quantitative CT renderings. Lung sections were subjected to staining (trichrome and H&E to assess collagen deposition as an indicator of pulmonary fibrosis. Whole lung homogenates were analyzed for total hydroxyproline and desmosine content (the latter not shown). Results are in Figures 11A -11D. All tests showed that CSP-4 inhibited BLM-induced pulmonary fibrosis. Consistent with the in vitro effects of both CSP and CSP-4 on FL fibroblasts from FPI lungs (see Figure 6D), CSP-4 exerted a In vivo beneficial effect against established pulmonary fibrosis, mimicking the beneficial effects of the full-length peptide, CSP (SEQ ID NO: 3).

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CSP-4 peptide and nutlin-3a inhibit FL fibroblast proliferation

Mice exposed to BLM 14 days before were treated with CSP-4 or its control (CP) or nutlin-3a IV (as in examples IX and X). Seven days later lung sections were obtained and subjected to IHC for Ki-67 to assess cell proliferation (see Figure 12A). The increased staining for ki-67 antigens observed in lung sections from fibrotic mice was significantly reduced by treatment with nutlin-3a or CSP-4. Fibroblasts isolated from the lungs of these animals (as described in Example X) were assayed for changes in the expression of proteins co I-I, p53 and later uPA and PAI-1 by immunoblotting and mRNA by quantitative RT-PCR (see Figures 12B-12C). FL fibroblasts from mice with established BLM-induced fibrosis showed increased expression of uPA and coI-I mRNA and proteins compared to NL fibroblasts from mice without lung injury. These cells also showed minimal expression of p53 and PAI-1. However, in fibroblasts from mice treated with nutlin-3a, the protein and mRNA expression of uPA and coI-I were significantly suppressed. These changes were associated with marked induction of p53 protein, and expression of PAI-1 protein and mRNA, indicating that the reestablishment of the p53-fibrinolytic system interplay mitigates fibrosis.

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Inhibition of fibrotic changes by ex vivo treatment with CSP-4 or nutlin-3a

Mice were exposed to intranasal BLM for 21 d to induce pulmonary fibrosis, sacrificed, and their lungs were excised and cut into small pieces and placed in culture medium. These tissue samples were thereafter treated with 10 nN CSP-4 peptide, control peptide (CP), or nutlin-3a for 72 h. Conditioned medium and tissue lysates were and analyzed for changes in coI-I and α-SMA by immunoblotting. The results (Fig. 13) showed that treatment of fibrotic lung tissue explants with CSP-4 or nutlin-3a inhibited fibrosis ex vivo. The full length CSP peptide gave similar inhibition of coI-I and α-SMA (results not shown). Nutlin-3a and CSP-4 are expected to act similarly to affect human IPF lung tissues ex vivo. The most recent data from the present inventors and recent articles (eg, Bhandary et al. Supra), strongly support the view that p53-mediated downstream changes in uPA and PAI-1 regulate fl fibroblast viability.

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Expression of p53 in FL fibroblasts and subsequent reversal of uPA fibrinolytic system mitigates pulmonary fibrosis in vivo

The results described herein and elsewhere (Bhandary YP et al., Am J Physiol: Lung Cell Mot Physiol 3O2: L463-473, 2012; Shetty SK et al., Am J Respir Cell Mol Biol 47: 474- 83, 2012) show that IP or IV injections with CSP or CSP-4 (Fig. 11A-11D), or IV or oral administration of nutlin-3a in wild-type (WT) mice (Fig. 1OA-1OD) starting 14 d after BLM mitigates established pulmonary fibrosis. Therefore, the mdm2-mediated degradation of the p53 protein and the loss of the subsequent p53-uPA system interrelation increases FL fibroblast viability and ECM production in vivo, leading to destruction of lung architecture and loss. of lung function. Since patients frequently present advanced stages of IPF at diagnosis, the mechanisms by which nutlin-3a and CSP-4 inhibit established pulmonary fibrosis, subsequent targets of nutlin-3a and CSP-4 are identified. These studies use lung tissues from IPF patients and mice with advanced pulmonary fibrosis due to BLM lung injury. The contribution and specificity of the p53-uPA system interplay will be confirmed using mice deficient in uPA, uPAR, PAI-1 and p53.

A. Determination of the effects of nutlin-3a and CSP-4 treatment in fibrosis using FL tissues (IPF) ex vivo

Freshly dissected FL lung tissues from IPF patients are treated with nutlin-3a (10 µM) or CSP-4 (10 nM) for 3 to 7 days (see Figure 13). Pulmonary tissues are homogenized and tested for changes in ECM (hydroxyproline and desmosine). Controls include fibrotic lung tissues (FL) from IPF patients treated with vehicle or control peptide, CP, or untreated lung tissues maintained in culture medium. Other controls include NL tissues excised from histologically "normal" lungs. The lungs of de-identified patients (“normal” and IPF) that were not suitable for transplantation and donated for medical research are obtained through IIAM (Edison, NJ) and the NDRI (Philadelphia, PA) and are used for ex studies. alive. Fibroblasts are isolated from these tissues and changes in p53, uPA, uPAR, and PAI-1 expression, ECM production, and viability are analyzed. Changes in the rate of ECM synthesis are also measured. FL tissue explants from IPF patients have elevated ECM synthesis when compared to expression levels in NL tissue sections from control subjects. However, FL lung explants treated with nutlin-3a or CSP-4 show significantly reduced MEC synthetic rates when compared to those that remain untreated or exposed to vehicle alone or CP. Fibroblasts isolated from FPI explants treated with nutlin-3a or CSP-4 show high levels of expression of p53, miR-34a and PAI-1 and reduced levels of uPA and uPAr. Akt phosphorylation and PDGFR-p mRNA and protein expression are also reduced in fibroblasts from FPI explants treated with nutlin-3a or CSP-4. The proliferation rate and ECM production of these fibroblasts are significantly lower than FL fibroblasts from untreated controls or treated with vehicle or CP, but comparable to fibroblasts isolated from NL tissues.

B. Role of p53 and m¡R-34a in the control of pulmonary fibrosis

Based on the above results, the lack of expression of p53 and m¡R-34a by FL fibroblasts is predicted to contribute to pulmonary fibrosis and that the reestablishment of basal expression of p53 by nutlin-3a or CSP-4 through the induction of MR-34a mitigates pulmonary fibrosis. Since maximum pulmonary fibrosis occurs between days 14-28 after BLM injury (Bhandary YP et al., Am J Physiol: Lung Cell Mol Physlol 302: L463-473, 2012; Bhandary and P et al., . Regulation of alveolar epithelial injury and lung fibrosis by p53-mediated changes in urokinase and plasminogen activator inhibitor-1. Am J Pathol ( submitted)) pre-m¡R-34a is expressed in mouse lung fibroblasts by IV injection of lentivirus ( LV) containing proa2 (l) collagen promoter with pre-m¡R-34a at 14 and 21 days after BLM lung injury. Pulmonary fibrosis is evaluated 28 days later. The role of m¡R-34a in nutlin-3a or CSP-4 mediated mitigation of pulmonary fibrosis is confirmed by expression of Lv of m¡R-34a-AS in fibroblasts using the proa2 (l) promoter in fibrotic lungs.

The role of p53 in the beneficial effects against pulmonary fibrosis is directly confirmed by expressing p53 in lung fibroblasts of mice with established fibrosis using LV or Ad-p53 containing collagen promoter proa (I) with or without inhibition of m¡R- 34a. Pulmonary fibrosis and m¡R-34a expression are assessed, eg, 28 days after BLM injury. Alternatively or in addition, p53 expression is inhibited using LV shRNA in WT mice with BLM-induced lung fibrosis or using p53-deficient mice with established lung fibrosis (Davis DW et al., J Exp Med 192: 857-869 , 2000). These mice are then challenged with nutlin-3a or CSP-4, eg, 14 and 21 d after BLM injury. Control mice with pulmonary fibrosis are challenged with non-specific shRNA or WT mice without challenge and treated with nutlin-3a or CSP-4. Mice are tested for pulmonary fibrosis and m¡R-34a expression, eg, 28 days after onset of BLM injury. The contribution of the p53-uPA system interrelation is confirmed by exposing WT and p53, uPA, uPAr and PAI-1 deficient mice to BLM for 14-21 d to induce pulmonary fibrosis, alter the expression of m¡R-34a in fibroblasts as described above, and testing for effects on fibrosis, eg, on day 28 post injury with BLM.

C. Restoration of p53 expression and subsequent p53-uPA fibrinolytic system interrelation by nutlin-3a or CSP-4 inhibits pulmonary fibrosis

The contribution of the p53-uPA fibrinolytic system interplay in mitigating lung fibrosis by nutlin-3a and CSP-4 in mice is confirmed by isolating fibroblasts from these mice, eg, 28 d after BLM injury. These cells are tested for changes in the expression of p53, uPA, uPa R and PAI-1 and production of ECM. It is expected to find that FL fibroblasts isolated from the lungs of BLM-injured mice will show elevated uPA and uPAR, viability and production of MEC. These FL fibroblasts show minimal p53 and PAI-1 expression compared to uninjured NL lung fibroblasts. However, fibroblasts from the lungs of mice with BLM-induced fibrosis exposed to nutlin-3a or CSP-4 show elevated p53. These cells are less proliferative with minimal ECM production. The expression of uPA and uPAR is reduced while PAI-1 increases compared to FL fibroblasts obtained from BLM-injured mice. The levels of uPA, uPAR, and PAI-1 in lung fibroblasts from mice treated with BLM nutlin-3a or BLM CSP-4 are comparable to NL fibroblasts isolated from uninjured mice.

To confirm the involvement of the p53-uPA system interplay, FL fibroblasts extracted from the lungs of BLM-injured mice are transduced with Ad-p53. BLM-injured lung fibroblasts exposed to nutlin-3a or CSP-4 are positive controls while those exposed to CP or Ad-EV are negative controls. Cells are tested for changes in uPA, uPAR, and PAI-1.

Ad-p53 transduction or treatment with nutlin-3a or CSP-4 increases the expression of PAI-1 and inhibits that of uPA and uPAR. These cells suppress ECM production and proliferation rate compared to FL fibroblasts exposed to CP or Ad-Ev. FL fibroblasts from BLM mice express markedly low levels of caveolin-1 and expression of caveolin-1 mitigates excess ECM production. Therefore, Fl fibroblasts are transduced with Ad-Cav-1 to see if p53 and subsequent p53-mediated changes in the uPA fibrinolytic system, ECM and cell viability are reduced compared to those treated with Ad-Ev.

D. Role of nutlin-3a or CSP-4 in inhibiting Akt phosphorylation in mitigation of pulmonary fibrosis

FL fibroblasts show high Akt phosphorylation and PDGFR-p expression that provides survival signals (Stambolic V et al. Mol Cell 8: 317-25, 2001; L, J et al. J Environ Pathol Toxic Oncol 23: 253- 66, 2004; Hoyle GW et al. Am J Pathol 154: 1763-75, 1999; Meinecke AK et al. Blood 14: 119 : 5931-42, 2012) In addition, p53 increases the expression of PTEN (Stambolic et al., supra; Mayo LD, et al. J Blot Chem 277: 5484-89, 2002) and inhibits PDGFR-p (Widau RC et al., Mol Cell Biol 32: 4270-82, 2012; Thornton JD et al., Cell Cycle 4: 1316-9, 2005) while PAI-1 inhibits Akt phosphorylation (Shetty SK et al., 2012, supra; Stambolic et al., Supra, Malinowsky K et al. Transí Oncol 5: 98-104, 2012) . Transduction of FL fibroblasts with Ad-Cav-1 inhibits Akt phosphorylation through increased PTEN activity (Tourkina E et al. Open Rheumatol J. 6: 116-22, 2012; Wang XM et al. J Exp Med 203: 2895-906, 2006; Xia H et al. Am J Pathol 176: 2626-37, 2010).

Claims (8)

re iv in d ic a tio ns 1. A pharmaceutical composition for use in treating a fibrotic condition, the composition comprises: (a) an administrable peptide or polypeptide consisting of the sequence FTTFTVT (SEQ ID NO: 1) and, optionally, wherein the administrable peptide or polypeptide is linked to, or associated with, an internalizing sequence; and (b) a pharmaceutically acceptable carrier or excipient. The composition for use of claim 1, formulated for pulmonary injection or instillation. 3. The composition for use of claim 1, formulated for pulmonary instillation. The composition for use of claim 1, wherein the administrable peptide or polypeptide consisting of the sequence Ft TFTVT (SEQ ID NO: 1) is linked to or associated with an internalizing sequence. The composition for use of claim 1, wherein the fibrotic condition comprises pulmonary fibrosis. 6. The composition for use of claim 1, wherein the fibrotic condition comprises idiopathic pulmonary fibrosis. The composition for use of claim 1, wherein the administrable peptide or polypeptide is composed of L-amino acids. The composition for use of claim 1, wherein the administrable peptide or polypeptide is protected at its N and C ends.